EFFICIENCY OF PHOTOAFFINITY LABELING DNA HOMOPOLYMERS AND COPOLYMERS WITH ETHIDIUM MONOAZIDE *

Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently-linked following photoactivation at the same drug/DNA input ratio. There were significant sequence-related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent-adduct formation decreased in the order poly(dG-dC)^. poly(dG-dC)> poly-(dG). poly(dC)poly(dA-dT). poly(dA-dT)poly(dA). poly(dT). Ethidium monoazide was about 2-fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G-C pairs than the A-T base counterparts. In low ionic buffers (0.015 M Na⁺), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40-fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.     

REPAIR SYNTHESIS IN HUMAN LYMPHOCYTES PROVOKED BY PHOTOLYSIS OF ETHIDIUM AZIDE

Abstract— The azide analog of ethidium was mixed with human lymphocytes and when photolyzed with visible light provoked repair synthesis as shown by incorporation of tritiated thymidine in the presence of hydroxyurea. The use of photolyzed drug, or incubation of drug-cell mixtures in the dark was without effect. These experiments should prove useful in targeting drug action sites and in studying the details of DNA repair.     

EXCISION REPAIR IN TETRAHYMENA: EVENTS FOLLOWING REFEEDING OF STARVED UV-IRRADIATED CELLS

Abstract —Starvation of early-log-phase Tetrahymena pyriformis in non-nutrient phosphate buffer for 24 h results in a 40 per cent increase in cell number, as well as a complete cessation of DNA synthesis. Low levels of DNA synthesis are detectable between 1 and 2h after starved cells are transferred to a nutrient medium. Larger amounts of DNA synthesis are detected after the first 2 h of refeeding, and one round of replication is complete 4.5 h after refeeding. Damage, caused by sublethal doses of UV radiation (254 nm) administered just prior to refeeding, to the DNA of starved Tetrahymena appears to be corrected by an excision-repair process after refeeding of starved, irradiated cells. Changes in buoyant density of DNA synthesized, rate of DNA synthesis, and the chromatographic distribution of photoproducts were investigated following refeeding of starved, irradiated cells. Excision repair begins 1 h after refeeding and appears to be essentially complete within 7 h. During this time, thymine dimers produced by irradiation are removed. Semiconservative DNA synthesis commences 2–3 h after the first appearance of excision repair. In addition, between 3 and 8 h after refeeding, the rate of DNA synthesis in irradiated, refed cultures is much lower than the rate of DNA synthesis in unirradiated, refed cultures. Also, the specific activity in vitro of DNA polymerase from irradiated refed cells is very much greater than that of polymerase from unirradiated, refed cells.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

IRRADIATION IN THE FROZEN STATE: A TECHNIQUE FOR DIRECT PHOTOAFFINITY LABELING

UV irradiation of rabbit muscle phosphofructokinase (PFK) in the presence of adenosine 3',5'-cyclic phosphate (cAMP) resulted in the covalent attachment of this ligand molecule to the enzyme protein. Irradiation in the frozen ice state enhanced the rate of this incorporation more than 10-fold above that achieved in aqueous solution, without significantly affecting the rate of photodestruction of the protein. [³H]-cAMP and [³²P]-cAMP were each incorporated into PFK at identical rates in the frozen state. Rates of photoincorporation in the frozen and liquid states were both half-maximal at a free ligand concentration approximately equal to the dissociation constant of cAMP and PFK. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP), both of which are known to compete for cAMP binding to PFK, inhibited photoincorporation of cAMP. Guanosine monophosphate (GMP), inosine monophosphate (IMP), and guanosine 3',5'-cyclic phosphate (cGMP), which do not compete for cAMP binding, had no effect on photoincorporation of cAMP. Irradiation of [³H]-AMP or [³H]-ADP resulted in photoincorporation into PFK at 0°C, with enhancement at — 77°C similar to that noted with cAMP.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

IS DNA TOPOISOMERASE INVOLVED IN THE UV EXCISION REPAIR PROCESS? NEW EVIDENCE FROM STUDIES WITH DNA INTERCALATING AND NON-INTERCALATING ANTITUMOR AGENTS

Abstract— The effects of selected DNA intercalating and non-intercalating drugs on the UV excision repair process in human fibroblasts have been examined. 9-Amino acridine, acridine orange, quinacrine, doxorubicin (adriamycin), ethidium bromide and actinomycin-D all inhibited the removal of pyrimidine dimers from cellular DNA by inhibiting the incision process as monitored by the nick translation assay and by an endonuclease-sensitive site assay. These agents also partially inhibited incision by the M. luteus endonuclease in an in vitro system. This is the only class of compounds tested to date that appears to block this early step of repair in mammalian cells. The DNA topoisomerase inhibitors, m -amsacrine and VP-16 (etoposide) and the bacterial gyrase inhibitors nalidixic acid and oxolinic acid were shown not to inhibit UV repair. As shown previously, however, novobiocin does block dimer removal and we show here that it is a potent inhibitor of the M. luteus UV endonuclease. While it has recently been demonstrated that many DNA intercalating agents block the strand-passing activity of DNA topoisomerase II giving rise to protein associated DNA strand breaks, the finding that the specific inhibitors of topoisomerase, m -AMSA and VP-16, do not inhibit repair, even though they block this strand passing activity, strongly suggests that inhibition of DNA topoisomerase is not associated with inhibition of DNA repair.     

DNA EXCISION REPAIR IN THE VERY UV-SENSITIVE XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP A STRAIN XP4LO

Abstract— XP4L0, a xeroderma pigmentosum complementation group A strain, exhibits very limited DNA repair activity. It has extreme sensitivity to UV (254 nm) as determined by colony forming ability. The rate of loss of UV (1 J/m²)-induced pyrimidine dimers from populations of quiescent, nondividing XP4LO cells was determined and found to be slower than that observed for other group A strains (XP25R0, XP12BE, XP8LO). The extreme UV-sensitivity is also exhibited by the nondividing cells in a survival assay that employs nondividing cell populations and does not involve cell reproduction. This result suggests that the extreme sensitivity measured previously by colony-forming ability (a cell-reproduction assay) is due to the excision repair defect alone and not to an additional post-replication repair defect. The very limited excision allows for an accurate definition of target size for inactivation of nondividing cells, about 1 pyrimidine dimer per 10⁵ base pairs, and when compared to results observed for other XP-A strains, provides further evidence that even though excision repair in group A is severely limited, it has biological significance.     

MORE EFFICIENT EXCISION REPAIR OF PYRIMIDINE DIMERS IN THE SPECIFIC DNA SEQUENCE THAN IN THE GENOME OVERALL IN GOLDFISH CELLS

Excision repair of pyrimidine dimers induced by 254 nm UV was examined in the genome overall and in a specific sequence containing a transfected gene for hygromycin B resistance, in RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. More than 40% of dimers were removed from the specific sequence, while about 20% were removed from the genome overall, within 24 h after exposure to UV (2.5-7.5 J/m2).     

THE TIME SEQUENCE OF EVENTS DURING EXCISION REPAIR IN TETRAHYMENA PYRIFORMIS

Abstract— The time sequence of events during excision repair of DNA in Tetrahymena pyriformis was investigated after sublethal dose of u.v. radiation. Buoyant-density analysis of the DNA from repairing cells grown in medium containing 5'-bromodeoxyuridine makes it possible to distinguish repair synthesis from normal synthesis. Analysis of the DNA synthesized at various times after irradiation clearly indicates that repair synthesis starts very quickly after irradiation and is completed within 3 to 4 hr. Immediately after irradiation, normal DNA synthesis is greatly depressed but gradually resumes until it predominates after 3 to 4 hr. The molecular weight of DNA strands is reduced and the net rate of DNA synthesis is depressed immediately after irradiation. Both of these parameters are restored to their pre-irradiation values by 3 to 4 hr after irradiation. During the repair period the majority of the pyrimidine dimers are removed. All of the data indicate that repair begins immediately after irradiation and is completed in 3 to 4 hr (about two thirds of a generation period).     

THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN p-BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY

Abstract— p -Benzoylphenylacetyl chymotrypsin, an acyl enzyme derivative containing the benzophenone group in the hydrophobic binding pocket, was prepared and is indefinitely stable at low pH. Photolysis of this covalent derivative leads to loss of enzymic activity and incorporation of the labeling group via formation of a second covalent bond. The efficiency of the photochemical processes is exceptionally high, producing 100% incorporation and at least 92% inactivation. Analysis of active site titration data for the photolyzed enzyme show that at least two different photochemical processes must be involved. Elimination of phosphorescence emission and reduction of UV absorption upon photolysis are consistent with initial hydrogen abstraction by benzophenone triplet state, followed by radical coupling, much as has been observed for the photoreaction of benzophenone with model systems. Photoaffinity labeling of chymotrypsin is also efficiently accomplished using two benzophenone derivatives which bind noncovalently to the enzyme's active site, although the rates of labeling are somewhat less than in the covalent complex.     

维多利亚蓝B标记分光光度法测定小牛胸腺DNA

研究了维多利亚蓝B(VBB)与小牛胸腺DNA(ctDNA)的相互作用。在室温、pH5.5的(CH2)6N4HCl条件下,ctDNA的加入使VBB在其最大吸收波长616nm处的吸光度明显下降,下降的程度与ctDNA的含量呈线性关系,线性范围为0～14μmol·L-1,检出限为(3σ)0.032mg·L-1,但在572nm波长处出现VBB ctDNA新的吸收峰,结合数为1∶1。以VBB为标记物,建立了一种测定ctDNA的新方法。方法具有操作简单、灵敏度和选择性较好。用于合成样品中ctDNA的分析,RSD为1.5%～2.1%,回收率为97.0%～98.2%,结果满意。     

ALPHA POLYMERASE INVOLVEMENT IN EXCISION REPAIR OF DAMAGE INDUCED BY SOLAR RADIATION AT DEFINED WAVELENGTHS IN HUMAN FIBROBLASTS

Abstract Cultured fibroblasts derived from normal human skin have been irradiated at a series of monochromatic wavelengths throughout the ultraviolet region and exposed to the specific α polymerase inhibitor, aphidicolin (1 μg/m l , 2 days) prior to assay for colony forming ability. Repair of 75-80% of the lethal damage induced by UVC (254 nm) or UVB (302 nm, 313 nm) radiation is inhibited by aphidicolin suggesting that such damage is repaired by a common α polymerase dependent pathway. Exposure to aphidicolin after irradiation at longer UVA (334 nm, 365 nm) or a visible (405 nm) wavelength leads to slight protection from inactivation implying that the processing of damage induced in this wavelength region is quite distinct from that occurring at the shorter wavelengths and does not involve α polymerase.     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

DNA REPAIR IN V-79 CELLS TREATED WITH COMBINATIONS OF PHYSICAL AND CHEMICAL CARCINOGENS*

Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair in Chinese hamster V-79 cells treated with 254 nm of ultraviolet radiation (UV), 7,12-dimethylbenz[a]anthracene 5,6-oxide (DMBA-epoxide), N-acetoxy-2-acetylaminofluorene (AAAF), 4-nitroquinoline 1-oxide (4NQO), 2-methoxy-6-chloro-9-[3(ethyl-2-chloroethyl)-aminopropylamino]acridine dihydrochloride (ICR-170), X-rays, ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS) and combinations of these agents. Compared to normal human cells V-79 were defective in repair of UV lesions and the lesions induced by the UV-mimetic chemicals. The extent of the defects varied from 10 to 50% and was similar to those in Xeroderma pigmentosum group C cells (XP C). V-79 cells repaired X-ray damage and damage from the alkylating agents EMS and MMS to the same extent as human cells. Repair was additive after a combination of UV plus MMS indicating, as expected, that there are different rate-limiting steps for removal of the damages from these agents. Repair was less than additive in cells treated with UV plus ICR-170, AAAF plus ICR-170, AAAF plus 4NQO, and 4NQO plus ICR-170 and approximately equal to that observed for the higher of the two agents separately, indicating that there may be similar rate-limiting steps for removal of lesions. Although the results on repair after combinations of UV plus 4NQO, UV plus DMBA-epoxide or X-rays plus MMS were difficult to interpret, there was not any inhibition of repair in these combinations.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

SATURATION OF DNA REPAIR IN MAMMALIAN CELLS*

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m². We measured excision repair in normal human fibroblasts up to 80J/m². The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus , and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m² doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.     

THE REPAIR OF DNA STRAND BREAKS IN HUMAN LYMPHOCYTES EXPOSED TO NEAR UV-RADIATION (UVA) AND FAR UV-RADIATION (UVC)

Abstract— UVA irradiation of human lymphocytes induces DNA strand breaks and a portion of these breaks are closed at a slower rate than X-ray induced DNA strand breaks and the strand breaks generated during repair of UVC induced DNA lesions. In addition, the yield of DNA strand breaks in lymphocytes pretreated with UVA radiation and given a subsequent exposure with UVC radiation is higher and shows a slower decrease with increasing repair time in comparison with the expected yield based on additivity between UVA and UVC induced DNA strand breaks. This indicates that UVA delays the closure of the intermediate strand breaks formed in the repair process of UVC induced DNA lesions.